PROJECT SUMMARY The myeloproliferative neoplasms (MPNs), which include polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are closely related clonal hematopoietic disorders that are characterized by extramedullary hematopoiesis, bleeding disorders, a shortened lifespan, and a propensity to evolve to AML. Currently there is no way to predict which patients will develop AML, and little is known about the genetic events that are associated with progression. The identification of drivers of leukemic transformation will improve our understanding of the disease and provide novel targets for therapeutic intervention. To define the pathways that drive AML progression, we performed a focused CRISPR/Cas9 screen to identify genes whose editing resulted in hematopoietic progenitor cell self-renewal of Jak2V617F cells but not wild-type progenitors. We identified STK11 and RPS6KA2 as two genes whose editing cooperates with the JAK2 mutant to promote transformation in vitro. Importantly we also found that STK11 and RPS6KA2 are downregulated and mutated in post-MPN AML, respectively, but not in chronic phase MPN. These results are highly innovative, provide significant insights into disease progression, and reveal two new pathways to development of AML. We hypothesize that alterations in STK11 induce AML by suppressing the activity of its substrates, including AMPK, and consequently altering cellular metabolism and protein translation, leading to increased expression of oncogenic proteins. Similarly, we hypothesize that genetic alterations in RPS6KA2 cause AML by impairing STK11 function and/or enhancing mTORC1 signaling and oncogenic protein translation. We propose to delve into the contributions of STK11 and RPS6KA2 to MPN progression by the following aims: 1) Investigate the mechanisms by which loss of STK11 induces progression of JAK2V617F mutant MPN to AML; and 2) Elucidate the contributions of alterations in RPS6KA2 to the progression of MPN to AML. Together these studies will yield important new insights into the genetic basis and mechanisms by which AML arises from the MPNs.